Aminoglycoside-resistant Streptococcus and Enterococcus species isolated from bovine mammary secretions.

A total of 117 isolates representing four Streptococcus species and 20 isolates representing two Enterococcus species from bovine mammary secretions were examined for resistance to streptomycin, kanamycin, and gentamicin. Resistance to streptomycin (85.4%) was most prevalent, followed by kanamycin (19%) and gentamicin (2.2%). Minimum inhibitory concentration of streptomycin for most organisms examined ranged from 16 to 250 micrograms/ml. For kanamycin, the minimum inhibitory concentration for most organisms was .5 to 62.5 micrograms/ml. Minimum inhibitory concentration for gentamicin was lower than for kanamycin. Two strains each of Streptococcus uberis and Enterococcus faecalis had minimum inhibitory concentration for streptomycin of greater than 2000 micrograms/ml, and two strains of E. faecalis had similar minimum inhibitory concentration for kanamycin. The minimum bactericidal concentration for all organisms was one to four times higher than minimum inhibitory concentration. None of the organisms evaluated was found to carry plasmids. Transfer of antibiotic resistance in plasmid-free strains was not achieved by mobilization with the aid of plasmid pAD1, indicating the absence of conjugative transposons. These data suggest that expression of aminoglycoside resistance of Streptococcus and Enterococcus species of bovine origin is likely due to factors other than plasmids or conjugative transposons.     

Phylogenetic studies on Corynebacterium bovis isolated from bovine mammary glands

Coryneform bacteria are frequently isolated from bovine mastitis with the lipophilic species, and Corynebacterium bovis is the most frequently isolated organism of this group. However, previous studies on the phylogeny of corynebacteria have incorporated only a single reference strain. We examined the phylogeny of C. bovis using 47 strains isolated from bovine mammary glands. Phylogenetic studies were performed by direct sequencing of the 16S ribosomal RNA and comparison to sequences of reference strains. All strains identified as C. bovis demonstrated similarity of 98% or higher to the ribosomal RNA gene sequences of the type strain of C. bovis. Phylogenetic analyses indicated that all strains tested clustered with members of the Corynebacterium urealyticum group confirming that C. bovis is a legitmate member of the genus Corynebacterium. Further investigation into the diversity within the species using repetitive element palindrome PCR indicated only minor differences between the strains tested. Corynebacterium bovis ATCC 13722 demonstrated the highest similarity (95%) with Brevibacterium helvolum, indicating that this organism does not belong in the genus Corynebacterium.     

A convenient method for differentiation of coagulase-negative staphylococci isolated from bovine mammary glands

The utility of trehalose-mannitol broth and arabinose-cellobiose broth for identification of Staphylococcus epidermidis and novobiocin-resistant staphylococci was determined using 236 coagulase-negative staphylococci isolated from bovine mammary glands. None of the 49 S. epidermidis strains was positive in trehalose-mannitol broth; whereas, all strains of Staphylococcus hyicus, Staphylococcus chromogenes, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus warneri, and Staphylococcus simulans were positive. Of the novobiocin-resistant staphylococcal species, only Staphylococcus saprophyticus was negative in arabinose-cellobiose broth. Except for one strain of Staphylococcus sciuri and one strain of Staphylococcus kloosii, all remaining strains of novobiocin-resistant staphylococcal species were positive in arabinose-cellobiose broth. Results indicate that trehalose-mannitol broth is an acceptable method for identification of S. epidermidis isolated from bovine mammary glands. Furthermore, arabinose-cellobiose broth is a useful method of screening for novobiocin-resistant staphylococci.     

Identification of corynebacterium bovis and other coryneforms isolated from bovine mammary glands

Bovine mastitis remains the most economically important disease in dairy cows. Corynebacterium bovis, a lipid-requiring Corynebacterium spp., is frequently isolated from the milk of infected mammary glands of dairy cows and is associated with reduced milk production. A total of 212 coryneform bacteria isolated from the milk of dairy cows were obtained from mastitis reference laboratories in the United States and Canada. All isolates had been presumptively identified as Corynebacterium bovis based on colony morphology and growth in the presence of butterfat. Preliminary identification of the isolates was based on Gram stain, oxidase, catalase, and growth on unsupplemented trypticase soy agar (TSA), TSA supplemented with 5% sheep blood, and TSA supplemented with 1% Tween 80. Of the 212 isolates tested, 183 were identified as Corynebacterium spp. based on preliminary characteristics. Of the strains misidentified, one was identified as a yeast, two as Bacillus spp., 11 as Enterobacteriaceae, 18 as staphylococci, one as a Streptococcus spp., and one as an Enterococcus spp. Eighty-seven coryneforms were selected for identification to the species level by direct sequencing of the 16S rRNA gene, the Biolog system and the API Coryne system. Fifty strains were identified as C. bovis by 16S rRNA gene similarity studies: the Biolog and API Coryne systems correctly identified 54.0 and 88.0% of these strains, respectively. The other coryneforms were identified as other Corynebacterium spp., Rhodococcus spp., or Microbacterium spp. These data indicate that the coryneform bacteria isolated from bovine mammary glands are a heterogeneous group of organisms. Routine identification of C. bovis should include Gram-stain, cell morphology, catalase production, nitrate reduction, stimulated growth on 1% Tween 80 supplemented media, and beta-galactosidase production as the minimum requirements.     

Genomic typing of enterococci isolated from bovine mammary glands and environmental sources

Enterococcal isolates (n = 102) from various sources of bovine origin on 1 farm were characterized using pulsed field gel electrophoresis analysis of SmaI restriction patterns. Isolates originated from feed samples (n = 6), bedding samples (n = 15), and bovine quarter-milk samples (n = 81). Isolates collected from milk samples included those from high-somatic cell count cows (n = 42), postpartum milk samples (n = 16), and clinical mastitis samples (n = 23). Species evaluated included Enterococcus faecium (n = 68), Enterococcus casseliflavus (n = 29), and Enterococcus faecalis (n = 5). A total of 20 clusters representing 44 isolates were detected when a similarity cut-off level of 75% was applied to interpret the pulsed field gel electrophoresis results. Fifteen of the clusters contained only isolates from milk samples. Four clusters contained isolates from bedding and milk samples. One cluster contained only isolates from feed samples. Clusters comprised of a single species represented 17 of the 20 total clusters. These results suggest enterococci from bovine origin were genetically diverse, whereas a limited number of isolates from various sources appeared to cluster together.     

Antimicrobial drug susceptibility of Staphylococcus aureus strains isolated from bovine and ovine mammary glands

The activity of selected antimicrobial agents against Staphylococcus aureus was determined with the agar disk diffusion test to determine the diameter of the zone of inhibition and the E-test for determination of the minimal inhibitory concentration (MIC). The 92 S. aureus strains used in this study were isolated from bovine (n = 76) and ovine (n = 16) intramammary infections. Four antibiotics, which are frequently used in mastitis therapy were chosen: penicillin-G, ampicillin, kanamycin, and cephalexine. The fifth compound (oxacillin) was used to detect methicillin-resistant S. aureus, but no such strains could be found. According to the evaluation criteria, 65.2 (penicillin) to 93.5% (kanamycin, cephalexine) S. aureus strains were susceptible to the antibiotics tested. Ovine S. aureus strains reveal a lower resistance rate than bovine isolates. Comparison of the results of the two methods of susceptibility testing shows, with exception of penicillin and ampicillin, satisfactory agreement. Analyzing the results of the MIC endpoints and the zone diameter values, very major errors, according to the error rate bounded method of Metzler and DeHaan, occurred at an error rate of 3.3% for penicillin and 3.8% for ampicillin.     

Plasmid pattern analysis of Staphylococcus species isolated from bovine mammary secretions.

Plasmid profiles of staphylococci isolated from bovine mammary secretions were heterogeneous as shown by the study of 94 isolates representing six species. Plasmids were identified in 19 of 94 staphylococcal isolates. Number of plasmids per isolate varied from 1 to 4. Size of plasmids ranged from 1.2 to 45 MDa; however, most were between 1.8 and 4.8 MDa. Some isolates with identical plasmid profiles were observed within and between species. Plasmid profiles observed in this study suggest that no specific plasmid pattern occurs within a species. Ability to differentiate isolates was not enhanced when antibiograms were used in conjunction with plasmid profiles. Plasmid pattern analysis does not appear to be an adequate method for discriminating between isolates of a species and would likely provide limited epidemiological information regarding staphylococci of bovine origin.     

Conventional identification of Streptococcus uberis isolated from bovine mastitis in Argentinean dairy herds

The objective of this study was to evaluate a conventional scheme for identifying Streptococcus uberis strains isolated from bovine mastitis. Seventy-five gram-positive, catalase-negative cocci were collected from cows with mastitis from 19 dairy herds located in the east-central region of Argentina. Five American Type Culture Collection strains and bovine isolates were identified by the API 20 Strep system and by restriction fragment length polymorphism analysis of 16S rDNA. A conventional scheme based on 11 biochemical tests was selected for identification of Strep. uberis strains: the Christie-Atkins-Munch-Petersen reaction; hydrolysis of Arg, esculin, and sodium hippurate; growth in inulin, mannitol, raffinose, salicin, and sorbitol; and growth at 45°C and in 6.5% NaCl. Reference strains and 25 bovine isolates were classified accurately to the species level by the conventional scheme in a blind assay. Each reference strain and each bovine isolate were identified as belonging to the same species following these 3 methods. The remaining 50 isolates identified as Strep. uberis by the API 20 Strep system and 16S rDNA RFLP were assayed by the conventional scheme. This scheme correctly identified 47 (94%) of 50 isolates as Strep. uberis by comparing their biochemical profile with that of the reference strain. Three (6%) of the 50 isolates were classified as Strep. uberis by the API 20 Strep system and by 16S rDNA RFLP and were identified as Enterococcus faecalis by the conventional scheme. Thirty percent of the Strep. uberis strains showed biochemical profiles identical to the Strep. uberis American Type Culture Collection 27958 strain. Seventy percent of the Strep. uberis strains demonstrated variability compared with the reference strain, resulting in 19 different biochemical profiles. The conventional scheme proposed in this study resulted in a relatively low number of misidentifications and could biochemically identify not only typical, but also atypical Strep. uberis strains. This conventional scheme can be considered an adequate method for identifying Strep. uberis strains isolated from bovine mastitis because of its affordable cost in developing countries, and it may contribute to determining the frequency of isolation of Strep. uberis strains in Argentinean dairy herds.     

Antimicrobial susceptibilities of staphylococcal species isolated from mammary glands of unbred and primigravid dairy heifers

Staphylococcal isolates from teat canal keratin and mammary secretion samples of unbred and primigravid Jersey heifers were tested in vitro for susceptibility to 12 antimicrobial agents. More than 92% of the 311 isolates were susceptible to all antimicrobial agents tested. Staphylococci other than Staphylococcus aureus demonstrated an overall susceptibility of 98.3% to all antibiotics, and Staphylococcus aureus demonstrated a 97% susceptibility. Across all staphylococcal species, susceptibility of isolates from secretion samples was 98.1%, and susceptibility of isolates from teat canal keratin samples was 93.1%. Differences in susceptibilities were observed among herds.     

Use of PCR-restriction fragment length polymorphism analysis for identification of yeast species isolated from bovine intramammary infection

This study reports a rapid PCR-based technique using a one-enzyme RFLP for discrimination of yeasts isolated from bovine clinical and subclinical mastitis milk samples. We analyzed a total of 1,486 milk samples collected over 1 yr in south Sardinia and northern Italy, and 142 yeast strains were preliminarily grouped based on their cultural morphology and physiological characteristics. Assimilation tests were conducted using the identification kit API ID 32C and APILAB Plus software (bioMérieux, Marcy l’Etoile, France). For PCR-RFLP analysis, the 18S-ITS1-5.8S ribosomal(r)DNA region was amplified and then digested with HaeIII, and dendrogram analysis of RFLP fragments was carried out. Furthermore, within each of the groups identified by the API or PCR-RFLP methods, the identification of isolates was confirmed by sequencing of the D1/D2 region using an ABI Prism 310 automatic sequencer (Applied Biosystems, Foster City, CA). The combined phenotypic and molecular approach enabled the identification of 17 yeast species belonging to the genera Candida (47.9%), Cryptococcus (21.1%), Trichosporon (19.7%), Geotrichum (7.1%), and Rhodotorula (4.2%). All Candida species were correctly identified by the API test and their identification confirmed by sequencing. All strains identified with the API system as Geotrichum candidum, Cryptococcus uniguttulatus, and Rhodotorula glutinis also produced characteristic restriction patterns and were confirmed as Galactomyces geotrichum (a teleomorph of G. candidum), Filobasidium uniguttulatum (teleomorph of Crypt. uniguttulatus), and R. glutinis, respectively, by D1/D2 rDNA sequencing. With regard to the genus Trichosporon, preliminary identification by API was problematic, whereas the RFLP technique used in this study gave characteristic restriction profiles for each species. Moreover, sequencing of the D1/D2 region allowed not only successful identification of Trichosporon gracile where API could not, but also correct identification of misidentified isolates. In conclusion, the 18S-ITS1-5.8S region appears to be useful in detecting genetic variability among yeast species, which is valuable for taxonomic purposes and for species identification. We have established an RFLP database for yeast species identified in milk samples using the software GelCompar II and the RFLP database constitutes an initial method for veterinary yeast identification.     

Serratia species isolated from bovine intramammary infections

Intramammary infections from which Serratia spp. were isolated were studied over a 32-mo period in a research dairy herd consisting of approximately 120 lactating cows. A total of 29 Serratia spp. intramammary infections were detected and accounted for 9% of all Gram-negative bacterial intramammary infections. Serratia marcescens was the most common Serratia spp. isolated. Origin of intramammary infections was 48.3% during the first half of the dry period, 31% during the last half of the dry period, and 20.7% during lactation. A total of 64% of intramammary infections that were first detected during the first half of the dry period persisted to calving. Geometric mean number of lactation days infected for all infections was 55. Intramammary infections that originated during the first half of the dry period were present in lactation for a significantly greater number of days compared with intramammary infections new during the last half of the dry period or lactation. A total of 48% of infections were clinical. Serratia spp. intramammary infections tended to be of long duration compared with other Gram-negative bacterial intramammary infections and were highly associated with the dry period.     

Functional activity of neutrophils from bovine mammary glands infected with Staphylococcus aureus

To test the effect of Staphylococcus aureus infection on mammary neutrophil function, intramammary neutrophils from S. aureus-infected quarters (n = 8), from adjacent uninfected quarters (n = 8) of S. aureus-infected cows, and from quarters (n = 8) of uninfected cows were collected and incubated with S. aureus in vitro. Mean percent neutrophils phagocytizing, number S. aureus per neutrophil, and log10 viable phagocytized S. aureus/ml were: 53.2, 6.4, and 4.72. Differences in function of neutrophils collected from infected and uninfected quarters were not statistically significant. Results indicate that function of neutrophils from S. aureus-infected quarters is similar to function of neutrophils from uninfected quarters.     

Evaluation of the Rapid Strep system for identification of gram-positive, catalase-negative cocci isolated from bovine intramammary infections

The Rapid Strep system (Analytab Products, Plainview, NY) was used to identify 199 gram-positive, catalase-negative cocci isolated from bovine intramammary infections. The system accurately identified 88.4% of isolates. The system identified 100% of 46 Streptococcus agalactiae, 100% of 48 Streptococcus dysgalactiae, 54.5% of 11 Streptococcus equinus, and 96.2% of 53 Streptococcus uberis isolates. Enterococcus spp. were identified correctly 83.3% of the time. One of 4 Streptococcus saccharolyticus strains was identified as Streptococcus bovis, the previous classification for this organism, and 8 Streptococcus equi ssp. equi strains were misidentified as Streptococcus dysgalactiae. The Rapid Strep system was determined to be an acceptable alternative to conventional methods for identification of gram-positive, catalase-negative cocci isolated from bovine intramammary infections.     

Identification and antimicrobial resistance of Streptococcus uberis and Streptococcus parauberis isolated from bovine milk samples

The conventional identification of Streptococcus uberis/parauberis group (n = 137) in clinical and subclinical bovine mastitis samples originating from 111 different farms was compared with identification based on 16 and 23S rRNA gene HindIII RFLP patterns used as operational taxonomic units in numerical analyses. On the basis of ribopattern analysis only 2 isolates belonged to S. parauberis, which is thus not a frequent cause of bovine intramammary infections in Finland. According to in vitro antimicrobial susceptibility testing, Streptococcus uberis is susceptible to β-lactam antibiotics. The prevalence of erythromycin (15.6%) and oxytetracycline (40.6%) resistance of clinical S. uberis isolates was higher than reported previously among subclinical isolates. The 2 subclinical S. parauberis isolates were susceptible to all the antimicrobials tested.     

Molecular typing of Streptococcus uberis strains isolated from cases of bovine mastitis

The discriminatory power of two polymerase chain reaction-based DNA fingerprinting methods, random amplified polymorphic DNA and repetitive extragenic palindrome were compared by subtyping 128 isolates of Streptococcus uberis cultured from cows in six different dairy herds in New Zealand. The typing results demonstrated that the majority of isolates possessed unique fingerprint profiles except on occasions where multiple isolates were obtained from individual cows. On these occasions, individual quarters of the mammary gland were generally, but not exclusively, infected by the same strain of bacteria. Both random amplified polymorphic DNA and repetitive extragenic palindromic typing assays were simple to perform, relatively inexpensive ($11.00 per reaction), and provided reliable and reproducible results. Furthermore, when these assays were used in conjunction with each other, they provided a means of confirmation of the specific DNA fingerprint patterns obtained.     

Transport of bovine milk xanthine oxidase into mammary glands of the rat

This research investigated transport of bovine milk xanthine oxidase into mammary glands of the lactating rat. Transport capability suggested an exogenous, nonmammary, source for the enzyme. Five lactating rats were injected intracardially with 100 microgram of purified iodine-125 labeled xanthine oxidase and five were injected with 100 microgram of the enzyme unpurified. Four hours later the rodents were hand-milked, and radiation was confirmed in all samples by liquid scintillation counting. Counts were recorded per volume of milk and the percentage radiation was computed. Autoradiographs of the rats indicated radiation almost exclusively associated with the mammary glands. Greatest concentration of radioactivity was in the micellar casein fraction of milk, and a compound of high molecular weight, presumably [iodine-125]xanthine oxidase, was confirmed by gel filtration of the casein. Results suggest that the compound was transported into the mammary glands. The degree of transport was dependent upon the stage of lactation.